Objectives: Traditional views of heterochromatin as transcriptionally repressive structures are being challenged by recent findings showing the existence of numerous essential protein-coding genes within heterochromatin and the implication of HPia and other heterochromatic factors in mediating transcriptional activation. The long-term objectives of this study are to determine the high-resolution higher-order chromatin organization of Drosophila heterochromatin and to elucidate the mechanisms regulating transcription and repression within this environment. Specific Aims: (i) Generation of high-resolution maps detailing chromatin composition and transcriptional activity of heterochromatin. (2) Analysis of the spatial arrangement of heterochromatic domains. (3) Identification and characterization of factors required for heterochromatin gene activation. Study Design: ChlP-chip technology and high-density tiling arrays will be used to compare the distributions of known heterochromatic and euchromatic factors on recently annotated heterochromatin domains. This will be used to analyze the role of heterochromatin proteins such as HPia on chromatin organization and gene activity within heterochromatin. In addition, a putative role for heterochromatin proteins in mediating long-range interactions of heterochromatic domains will be tested. A more direct role on gene expression will also be explored through characterization of HPia recruitment to heterochromatin genes and identification of factors that govern heterochromatin gene expression. This will be conducted through purification of native HPia complexes and genome-wide, RNAi-based screening of factors that contribute to heterochromatin gene expression. Relevance: This study will have a broad impact on our understanding of genome regulation by providing a more comprehensive and accurate model of Drosophila heterochromatin structure and gene function, which shares many similarities with mammalian genomes. Moreover, the central role of heterochromatin factors in genome stability, developmental regulation, and euchromatic gene expression, which include tumor suppressor pathways, has important implications to human biology and health.